Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the 'ECoScreen' in healthy calves (body weight 63-98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system 'ECoCheck'. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138-624 nmol l^-1 EBC or 0.10-0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC-H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC-pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC-pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

Fractionated breath condensate sampling: H2O2 concentrations of the alveolar fraction may be related to asthma control in children

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Increase of pro-oxidants with no evidence of lipid peroxidation in exhaled breath condensate after a 10-km race in non-athletes

It is a well-established fact that exercise increases pro-oxidants and favors oxidative stress; however, this phenomenon has been poorly studied in human lungs. Pro-oxidative generation (H₂O₂, NO₂ ^?), lipid peroxidation markers (MDA), and inflammation (pH) in exhaled breath condensate (EBC) have been determined through data from 10 active subjects who ran 10 km; samples were obtained immediately before, at 20, and at 80 min post-exertion. In EBC, the concentration of H₂O₂ at 80 min post-exertion was increased. NO₂ ^? concentration showed a tendency to increase at 80 min post-exertion, with no variations in MDA and pH. No variations of NO₂ ^? were found in plasma, while there was an increase of NO₂ ^? at 80 min post-exertion in the relation between EBC and plasma. NO₂ ^? in EBC did not correlate to plasmatic NO₂ ^?, while it did correlate directly with H₂O₂ in EBC, suggesting a localized origin for the exercise-related NO₂ ^? increase in EBC. MDA in plasma did not increase nor correlate with MDA in EBC. In conclusion, high-intensity exercise increases lung-originated pro-oxidants in non-athlete subjects with no evidence of early lipid peroxidation and changes in the pH value in EBC.     

Breath Condensate Hydrogen Peroxide Correlates with Both Airway Cytology and Epithelial Lining Fluid Ascorbic Acid Concentration in the Horse

The relationship between hydrogen peroxide (H²O²) concentration in expired breath condensate (EBC) and cytology of the respiratory tract obtained from tracheal wash (TW) or bronchoalveolar lavage (BAL), and epithelial lining fluid (ELF) antioxidant status is unknown. To examine this we analysed the concentration of H²O² in breath condensate from healthy horses and horses affected by recurrent airway obstruction (RAO), a condition considered to be an animal model of human asthma. The degree of airway inflammation was determined by assessing TW inflammation as mucus, cell density and neutrophil scores, and by BAL cytology. ELF antioxidant status was determined by measurement of ascorbic acid, dehydroascorbate, reduced and oxidised glutathione, uric acid and α-tocopherol concentrations. RAO-affected horses with marked airway inflammation had significantly higher concentrations of breath condensate H²O² than control horses and RAO-affected horses in the absence of inflammation (2.0±0.5?μmol/l, 0.4±0.2?μmol/l and 0.9±0.2?μmol/l H²O², respectively; p<0.0001). The concentration of breath condensate H²O² was related inversely to the concentration of ascorbic acid in ELF (r=-0.80; p<0.0001) and correlated positively with TW inflammation score (r=0.76, p<0.0001) and BAL neutrophil count (r=0.80, p<0.0001). We conclude that the concentration of H²O² in breath condensate influences the ELF ascorbic acid concentration and provides a non-invasive diagnostic indicator of the severity of neutrophilic airway inflammation.     

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Evaluation of methodological and biological influences on the collection and composition of exhaled breath condensate

Abstract

The purpose of this inter-species comparison (calves and pigs) was to identify methodological and biological influences on the collection and composition of exhaled breath condensate (EBC). A total of 352 EBC samples were collected, whilst variables of ventilation were registered in parallel. Partial pressure of carbon dioxide (pCO₂) and pH were analysed in non-degassed EBC samples. The concentration of total protein in EBC was measured colorimetrically. In both species, lung function was evaluated before and after EBC collection. Statistical analyses were performed to study the effect of EBC collection on lung function and to identify the influence of ventilatory variables on the collection and composition of EBC. Collection of EBC did not affect lung function. Despite the volume of EBC collected per unit time being primarily dependent on ventilation per unit time, species-specific conditions during the EBC collection process resulted in different dependences of EBC collection from other variables of ventilation (i.e. maximal airflow during expiration or expired tidal volume kg^?1 body weight). The concentration of protein ml^?1 EBC increased with the expired volume per min and with peak expiratory flow. Although the pCO₂ in fresh EBC was significantly negatively dependent on the duration of collection, comparable pHs (5.6???6.2) were measured in EBC of both calves and pigs. The obtained data may help one standardize EBC collection in different species.     

Optimising hydrogen peroxide measurement in exhaled breath condensate

Abstract

Background: Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H₂O₂), have been measured in EBC, without adequate published details of validation and optimisation.

Objectives: To explore factors that affect accurate quantitation of H₂O₂ in EBC.

Materials and Methods: H₂O₂ was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature.

Results: Standard curve slope was significantly altered by pH, leading to a potential difference in H₂O₂ quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H₂O₂ added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H₂O₂ ranged from 3.4 nM to 8.8 nM depending on the buffer used.

Conclusions: The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H₂O₂ in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H₂O₂ in EBC requires rigorous optimisation.     

Exhaled breath condensate as matrix for toluene detection: A preliminary study

Abstract

The study was designed to investigate whether exhaled breath condensate, obtained by cooling exhaled air in spontaneous breathing, could be a suitable matrix for toluene quantitative analyses. Nine healthy subjects were exposed for a short period (20 min) to a known concentration of toluene. Exhaled breath condensate samples were collected before and at the end of the exposure, while the environmental concentration of toluene was continuously monitored. Toluene was analysed by head-space gas-chromatography mass spectrometry, and assay repeatability was also estimated in vitro. Baseline and post-exposure measurement of hippuric acid, the urinary toluene metabolite, was performed to assess current toluene exposure. Before the exposure toluene concentrations in the exhaled breath condensate were lower than the detectable limit in all subjects, while after the exposure toluene was detectable with a median value 0.35 µg l^?1 (range 0.15–0.55 µg l^?1) in all the exhaled breath condensate samples. As compared with the standard calibration in distilled water, the curves obtained by exhaled breath condensate were linear and comparable with the range examined in vivo for toluene. A significant correlation was found between the environmental toluene levels and toluene in the exhaled breath condensate at the end of exposure. Furthermore, a significant relationship between increased exhaled breath condensate toluene levels and urinary hippuric acid after the exposure was found. In conclusion, exhaled breath condensate is a promising matrix for toluene assessment, although its application in humans requires further investigations.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

Partitioning of photosynthetic electron flow between CO2 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.) at different CO2 concentrations and during drought stress

Photosystem II chlorophyll fluorescence and leaf net gas exchanges (CO₂ and H₂O) were measured simultaneously on bean leaves (Phaseolus vulgaris L.) submitted either to different ambient CO₂ concentrations or to a drought stress. When leaves are under photorespiratory conditions, a simple fluorescence parameter F/ F_m (B. Genty et al. 1989, Biochem. Biophys. Acta 990, 87–92; F = difference between maximum, F_m, and steady-state fluorescence emissions) allows the calculation of the total rate of photosynthetic electron-transport and the rate of electron transport to O₂. These rates are in agreement with the measurements of leaf O₂ absorption using ¹⁸O₂ and the kinetic properties of ribulose-1,5bisphosphate carboxylase/oxygenase. The fluorescence parameter, F/F_m, showed that the allocation of photosynthetic electrons to O₂ was increased during the desiccation of a leaf. Decreasing leaf net CO₂ uptake, either by decreasing the ambient CO₂ concentration or by dehydrating a leaf, had the same effect on the partitioning of photosynthetic electrons between CO₂ and O₂ reduction. It is concluded that the decline of net CO₂ uptake of a leaf under drought stress is only due, at least for a mild reversible stress (causing at most a leaf water deficit of 35%), to stomatal closure which leads to a decrease in leaf internal CO₂ concentration. Since, during the dehydration of a leaf, the calculated internal CO₂ concentration remained constant or even increased we conclude that this calculation is misleading under such conditions.Abbreviations Ca, Ci ambient, leaf internal CO₂ concentrations - F_m, F_o, F_s maximum, minimal, steady-state fluorescence emission - F_v variable fluorescence emission - PPFD photosynthetic photon flux density - q_p, q_N photochemical, non-photochemical fluorescence quenching - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Multiple reactions in vanadyl-V(IV) oxidation by H2O2

Oxidation of vanadyl sulfate by H₂O₂ involves multiple reactions at neutral pH conditions. The primary reaction was found to be oxidation of V(IV) to V(V) using 0.5 equivalent of H₂O₂, based on the loss of blue color and the visible spectrum. The loss of V(IV) and formation V(V) compounds were confirmed by ESR and⁵¹V-NMR spectra, respectively. In the presence of excess H₂O₂ (more than two equivalents), the V(V) was converted into diperoxovanadate, the major end-product of these reactions, identified by changes in absorbance in ultraviolet region and by the specific chemical shift in NMR spectrum. The stoichiometric studies on the H₂O₂ consumed in this reaction support the occurrence of reactions of two-electron oxidation followed by complexing two molecules of H₂O₂. Addition of a variety of compounds—Tris, ethanol, mannitol, benzoate, formate (hydroxyl radical quenching), histidine, imidazole (singlet oxygen quenching), and citrate—stimulated a secondary reaction of oxygen-consumption that also used V(IV) as the reducing source. This reaction requires concomitant oxidation of vanadyl by H₂O₂, favoured at low H₂O₂:V(IV) ratio. Another secondary reaction of oxygen release was found to occur during vanadyl oxidation by H₂O₂ in acidic medium in which the end-product was not diperoxovanadate but appears to be a mixture of VO ₃ ⁺ (–546 ppm), VO³⁺ (–531 ppm) and VO ₂ ⁺ (–512 ppm), as shown by the⁵¹V-NMR spectrum. This reaction also occurred in phosphate-buffered medium but only on second addition of vanadyl. The compounds that stimulated the oxygen-consumption reaction were found to inhibit the oxygen-release reaction. A combination of these reactions occur depending on the proportion of the reactants (vanadyl and H₂O₂), the pH of the medium and the presence of some compounds that affect the secondary reactions.     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

The biogenesis of [Fe–S] clusters: the role of the unorthodox ABC ATPase,SufC, and the wider implications for understanding iron metabolism

Abstract

Oxidative stress is the hallmark of various chronic inflammatory lung diseases. Increased concentrations of reactive oxygen species (ROS) in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patient's discomfort. As a consequence, there is a need for less or non-invasive approaches to measure oxidative stress. The collection of exhaled breath condensate (EBC) has recently emerged as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. The biomarkers of oxidative stress such as H₂O₂, F₂-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species have been successfully measured in EBC. The reproducibility, sensitivity and specificity of the methodologies used in the measurements of EBC oxidative stress biomarkers are discussed. Oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. EBC is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. It is expected that EBC can be exploited to detect specific levels of biomarkers and monitor disease severity in response to appropriate prescribed therapy/treatment.     

Hemolymph oxygen content,oxyhemocyanin, protein levels and ammonia excretion in the shrimp Penaeus monodon exposed to ambient nitrite

Subadult Penaeus monodon (21.03±3.19 g) were exposed individually in sea water (30 mg·ml^-1) to 0.02 (control), 1.04, 5.02, 10.11 and 20.06 mg·l^-1 nitrite-N for 24h. Hemolymph pH, partial pressures of oxygen and carbon dioxide, bicarbonate concentration, oxyhemocyanin and protein levels, and whole animal ammonia-N excretion and nitrite-N uptake were determined. Ammonia-N excretion and hemolymph oxygen partial pressure increased, whereas hemolymph pH, HCO ₃ ^- , oxyhemocyanin, protein and the ratio of oxyhemocyanin/protein levels decreased with increasing ambient nitrite-N. It is suggested that accumulated nitrite of P. monodon following exposure to ambient nitrite causes reduction of oxyhemocyanin, protein and the ratio of oxyhemocyanin/protein in the hemolymph, and affects nitrogen metabolism and acid-base balance at low hemolymph pH.Abbreviations bw body weight - EC₅₀ concentration reducing growth rate by 50% that of controls - LC₅₀ median lethal concentration - nitrite-N nitrite concentration measured as nitrogen - PO₂ partial pressure of O₂ in hemolymph - PCO₂ partial pressure of CO₂ in hemolymph - sw sea water - ww wet weight     

Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands

Hydrogen peroxide (H₂O₂)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C. Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca²⁺ ions were not required but Ca²⁺ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H₂O₂ concentrations but only extent of aggregation at optimal H₂O₂ concentrations. Ba²⁺, Sr²⁺, Cd²⁺, Mn²⁺ and Ni²⁺ ions (1 mM) and Zn²⁺, Pb²⁺ and Hg²⁺ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the prostaglandin H₂/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane A₂, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein kinase C modulator, Zn²⁺ ions, inhibited by different mechanisms. The results indicate direct action of H₂O₂ at the prostaglandin H₂/thromboxane A₂ receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products. The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation.     

H2O2 Sensors of Lungs and Blood Vessels and Their Role in the Antioxidant Defense of the Body

This paper considers the composition and function of sensory systems monitoring H₂O₂ level by the lung neuroepithelial cells and carotid bodies. These systems are localized in the plasma membrane of the corresponding cells and are composed of O ² -generating NADPH-oxidase and an H₂O₂-activated K⁺ channel. This complex structure of the H₂O₂ sensors is probably due to their function in antioxidant defense. By means of these sensors, an increase in the H₂O₂ level in lung or blood results in a decrease in lung ventilation and constriction of blood vessels. This action lowers the O₂ flux to the tissues and, hence, intracellular [O₂]. The [O₂] decrease, in turn, inhibits intracellular generation of reactive oxygen species. The possible roles of such systems under normal conditions (e.g., the effect of O ² in air) and in some pathologies (e.g., pneumonia) is discussed.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.